Compositions for culturing spirochetes

ABSTRACT

The present invention relates to methods and compositions for culturing spirochetes and treating spirochetal diseases. For example, the present invention provides serum-free media for culturing spirochete bacteria in vitro. The present invention further provides methods for identifying spirochete susceptibilities to antimicrobials and antimicrobial compositions and cocktails. The present invention also provides methods for treating subjects suspected of having a spirochete infection.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for culturingspirochetes and treating spirochetal diseases. For example, the presentinvention provides serum-free media for culturing spirochete bacteria invitro. The present invention further provides methods for identifyingspirochete susceptibilities to antimicrobials and antimicrobialcompositions and cocktails. The present invention also provides methodsfor treating subjects suspected of having a spirochete infection.

BACKGROUND OF THE INVENTION

Members of the Order Spirochaetales include organisms responsible fordiseases associated with significant morbidity and mortality in humansand other animals. The spirochetes are helically shaped, motile bacteriathat stain as Gram negative, and include the genera Borrelia,Brachyspira, Cristipira, Leptonema, Leptospira, Serpulina, Spirochaeta,Treponema, Pillotina, Diplocalyx, Hollandina, and Clevelandina. Of thesegenera, the Borrelia, Leptospira, and Treponema are responsible for themajority of human disease.

The Spirochetes

The genus Borrelia includes numerous species, the most clinicallysignificant of which is B. burgdorferi, the etiologic agent of Lymedisease. Other Borrelia are historically significant as causative agentsof epidemics, including B. recurrentis (synonyms include B. obermeyeriand B. novyi), B. duttoni, B. hermisii, and various others. For example,during the first half of the 1900s, more than 50 million peoplecontracted louse-borne relapsing fever, with epidemics occurringthroughout Europe, Africa, Asia, and South America (Schwan et al.,Borrelia, in Murray (ed.), Manual of Clinical Microbiology, AmericanSociety for Microbiology, Washington, D.C. [1995], pages 626-635). Since1959, more than 840 cases, including at least 6 deaths have beenrecorded from Jordan, Rwanda and Iran (See, Schwan et al., supra).

The genus Leptospira includes pathogenic as well as non-pathogenicserovars, although the pathogenicity is not a criterion for speciesdifferentiation. Nonetheless, the pathogenic serovars have beentraditionally included within the species L. interrogans and thefree-living non-pathogenic serovars have been included within thespecies L. biflexa. Over 210 serovars of L. interrogans and 63 serovarsof L. biflexa have been officially described (Kaufmann and Weyant,Leptospiraceae, in Murray (ed.), Manual of Clinical Microbiology,American Society for Microbiology, Washington, D.C. [1995], pages621-625). Leptospirosis is a zoonotic disease, with reservoirs in wild,domestic and feral animals. The disease can be very serious in humansand other animals, most notably pinnipeds. In humans, it ischaracterized by a biphasic illness in which about 10% of patientsdevelop icteric leptospirosis. Icteric leptospirosis can be clinicallysevere, with a mortality rate of approximately 10% (See, Kaufmann andWeyant, supra).

The genus Treponema includes four human pathogens and at least sixspecies that are not human pathogens. The most clinically significantspecies is T. pallidum, which includes three subspecies. T. pallidumsubsp. pallidum is the etiologic agent of venereal syphilis, while T.pallidum subsp. pertenue is the etiologic agent of yaws (frambesia,pian), and T. pallidum subsp. endemicum is the etiologic agent ofendemic syphilis (bejel, dichuchwa). T. carateum, the etiologic agent ofpinta (carate, cute) represents the fourth pathogenic organism withinthis genus. Despite the availability of effective therapy, venerealsyphilis remains an important sexually transmitted disease with aworldwide distribution. For example, in the United States, 112,581 caseswere reported in 1992, which included 33,973 cases of primary andsecondary syphilis and 3,850 cases of congenital syphilis (Norris andLarsen, Treponema and Other Host-Associated Spirochetes Murray (ed.),Manual of Clinical Microbiology, American Society for Microbiology,Washington, D.C. [1995], pages 636-651). Although venereal syphilis isof greatest concern in the United States, the other forms of treponemaldisease are significant in terms of morbidity and mortality worldwide.Endemic syphilis is restricted to the desert and temperate regions ofNorth Africa and the Middle East, while yaws occurs most frequently inthe tropical and desert regions of Africa, South America, and Indonesia,and pinta is primarily observed in tropical areas of Central and SouthAmerica (Norris and Larsen, supra). Yaws, endemic syphilis and pintawere endemic in certain areas prior to the establishment of eradicationprograms by the World Health Organization. In the 1950s, it wasestimated that 200 million people were exposed to these diseases (Norrisand Larsen, supra).

Cultivation and Identification of Spirochetes

In general, the spirochetes are difficult to culture and some have arequirement for in vivo cultivation methods. Indeed, Lyme diseasespirochetes are difficult to detect in human patients. However, Borreliacan be cultivated either in their arthropod vectors or in a largevariety of vertebrate hosts, although cultivation in embryonated chickeneggs is also possible. In vitro cultivation of Borrelia is most oftendone using Barbour-Stoenner-Kelly II (BSK II) medium, pH 7, withincubation in a microaerophilic environment at 30° to 37° C. The culturemedium is monitored for spirochetes by dark field microscopy for 4 to 6weeks. Traditionally, cultivation is conducted in the presence ofgelatin and rabbit serum, as it has been reported that many months ofincubation may be required to successfully grow these organisms in theabsence of these components (See, Schwan et al., supra). An additionalconsideration is that continuous serial passages, even over a shortperiod may effect many biological changes in the organisms, altering thephenotypic and genotypic characteristics.

Historically, identification of the Borrelia heavily depended upon thegeographic distribution and natural arthropod vectors. The developmentof molecular diagnostic procedures has greatly enhanced the ability toidentify these organisms, although direct identification of B.burgdorferi in clinical material by polymerase chain reaction (PCR) hasproven unreliable, except in cases where synovial fluid is used (See,Schwann et al., supra). Serologic confirmation of borrelioses is oftenattempted using methods such as immunofluorescence (IFA) and enzymeimmunoassays (ELISA or EIA), and Western blots. In addition, molecularidentification methods have been developed.

Disease caused by leptospires is often presumptively diagnosed based ondirect detection of organisms in a sample. These methods require skilland experience in order to correctly differentiate organisms fromartifacts in the samples. In vitro methods have also been developed tocultivate leptospires. However, their fastidious nature makes this acomplicated and time-consuming undertaking. Most often, semisolid mediumsuch as Fletcher's, Ellinghausen's, or polysorbate 80 is used.Traditionally, leptospiral cultures are maintained at room temperature,and cultures are observed once a week, for at least five weeks, usingdark field microscopy. Before reporting a culture as “negative,” theculture is examined twice a month for four months. Once grown inculture, leptospires can be identified to serogroup by the microscopicagglutination test (MAT). Other serologic methods have also beendeveloped (e.g., indirect hemagglutination, slide agglutination, andELISA), although none of these alternative methods appear to have thenecessary sensitivity and specificity for clinical diagnostics (See,Kaufmann and Weyant). The Treponema present even greater challenges tothe microbiologist, as the T. pallidum subspecies and T. carateum areobligate human parasites, with no known non-human animal orenvironmental reservoirs. Thus, diagnosis of such diseases as syphilisis based on direct microscopic examination of material collected fromlesions, non-treponemal tests (for screening), and treponemal tests (forconfirmation). The criteria for syphilis diagnosis are divided intothree categories, namely definitive, presumptive, and suggestive. TheTreponemes are reported as noncultivatable in vitro. In the UnitedStates, the routine testing scheme is direct microscopic examination oflesion exudates, followed by a non-treponemal test, which is thenconfirmed with a treponemal test, if positive (See, Norris and Larsen).The diagnosis of other treponemal diseases is even more cumbersome, asno laboratory methods have been developed to distinguish the otherpathogenic treponematoses from each other or from syphilis. Indeed, thestandard serologic tests for syphilis are uniformly reactive with yaws,pinta, and endemic syphilis (See, Norris and Larsen). Thus, diagnosis ofthese diseases can be problematic.

Treatment of Spirochetal Diseases

For borrelial infections, there appears to be general agreement thatantibiotics are preferable to the arsenical compounds traditionally usedto treat these diseases. For Lyme disease, treatment regimens dependupon the nature and severity of the clinical manifestations. Very fewprospective randomized therapeutic trials have been conducted, and MICs(minimum inhibitory concentrations) and MBCs (minimum bactericidalconcentrations) are inconsistently reported in the literature.

For leptospires, standardized procedures have yet to be developed forantimicrobial susceptibility testing (See, Kaufmann and Weyant).However, in vitro testing has demonstrated strain variability in thesusceptibility of the organisms to penicillin and tetracyclines. Indeed,additional methods need to be developed before in vitro testing ofleptospires can be recommended for selection of treatment regimens.Furthermore, the limited availability of laboratories with the requisitecapabilities for leptospiral disease diagnostics remains a problemworldwide.

In the United States, the Centers for Disease Control (CDC) publishrecommended guidelines for the treatment of syphilis. Treatment ofsyphilis is often empirical, as antimicrobial testing is notstraightforward (i.e., due to the lack of a method for continuousculture of T. pallidum). Various approaches been developed to determinethe susceptibilities of representative strains (e.g., Nichols strain) toantimicrobial agents based on criteria such as the in vitro loss ofmobility or infectivity, treatment of experimental animal infections,human trials, and examination of non-pathogenic cultivable treponemes.

SUMMARY

In sum, methods and compositions are needed for the reliablecultivation, detection, identification, and treatment of spirochetaldisease. The currently used methods are very cumbersome, time-consuming,and require a high level of skill and experience to perform. The needfor improved methods and compositions is highlighted by the increase indiseases associated with spirochetes.

Furthermore, the lack of reliable, easy to use culturing methods alsohas led to an unfortunate lack of information regarding the many diseasestates that may have an underlying spirochete component, and that may betreated with antibiotics specific for the spirochete. What is needed arereliable methods for culturing all spirochetes. The art is also in needof effective treatments for spirochete infections and methods fordetecting spirochete infection and detecting diseases associated withspirochete infection.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for culturingspirochetes and treating spirochetal diseases. For example, the presentinvention provides serum-free media for culturing spirochete bacteria invitro. The present invention further provides methods for identifyingspirochete susceptibilities to antimicrobials and antimicrobialcompositions and cocktails. The present invention also provides methodsfor treating subjects suspected of having a spirochete infection.

The present invention provides a composition comprising culture mediacapable of growing Treponema organisms in vitro. In some embodiments ofthe present invention the composition is capable of growing Treponemaorganisms in vitro under microaerophilic conditions (i.e., underconditions containing oxygen, but at a lower than atmosphericconcentrations). In other embodiments of the present invention, thecomposition is capable of growing Treponema organisms in vitro underanaerobic conditions (i.e., absence of oxygen). In some embodiments, thecomposition further comprising a dividing population of Treponemaorganism. In preferred embodiments, the Treponema organisms compriseTreponema pallidum. In some embodiments, the culture media is serum-freeculture media.

The present invention also provides a composition comprising serum-freeculture media capable of growing Treponema, Borrelia, and Leptospiraorganisms (i.e., capable of growing each of the organisms). In someembodiments of the present invention, the serum-free culture media iscapable of growing Treponema, Borrelia, and Leptospira organisms invitro. In some embodiments, the composition further comprises a dividingpopulation of a spirochete organism.

The present invention further provides methods for detecting spirochetesin a sample comprising providing: a sample suspected of containing atleast one genus of spirochete organism, and serum-free culture mediacapable of growing Treponema, Borrelia, and Leptospira organisms invitro; inoculating the culture media with the sample; culturing thesample in the culture media to produce an expanded culture; anddetecting the presence of the spirochete organisms in the expandedculture. The present invention is not limited to any particular sampletype. In some embodiments, the sample is selected from a water sample,an animal sample, an insect sample, and a fluid sample. In preferredembodiments, the animal sample comprises a tissue or fluid. In preferredembodiments, the animal sample comprises a human sample. In particularlypreferred embodiments, the human sample comprises a sample from apatient suspected of having a neurological or autoimmune disease. Forexample, in some embodiments, the human sample comprises a sample from apatient having symptoms of a disease selected from multiple sclerosisand rheumatoid arthritis. In preferred embodiments, the spirocheteorganisms is selected from Treponema, Borrelia, and Leptospira.

The present invention also provides a method for determiningantimicrobial susceptibility of spirochete organisms, comprisingproviding: a sample suspected of having a spirochete organism, culturemedia capable of growing Treponema, Borrelia, and Leptospira organismsin vitro; and one or more antimicrobial agents; culturing the sample inthe culture media to produce an expanded culture; treating the expandedculture with the one or more antimicrobial agents; and determining thesusceptibility of the spirochete organisms in the expanded culture tothe one or more antimicrobial agents. In preferred embodiments, thespirochete organisms is selected from Treponema, Borrelia, andLeptospira. In some embodiments, the one or more antimicrobial agentscomprise antibiotics. In some embodiments, the sample comprises a samplefrom a human patient, wherein the method further comprises the steps ofselecting one or more antimicrobials that kill the spirochetes; andtreating the human patient with the one or more antimicrobials that killthe spirochetes.

In yet other embodiments, the further comprising the step of selectingone or more antimicrobials that kill said spirochetes. The presentinvention further provides an antimicrobial composition comprising atleast one antimicrobial agent selected using the above methods.

The present invention also provides an antimicrobial compositioncontaining one or more antimicrobial agents selected using a methodcomprising: providing a plurality of samples containing at least onespirochete organism; conducting an in vitro antimicrobial susceptibilityassay on the plurality of samples; and selecting one or moreantimicrobial agents collectively capable of killing spirochetes in atleast 80% of said samples. In preferred embodiments, the antimicrobialagents are collectively capable of killing spirochetes in at least 90%of the samples, more preferably at least 95% of the samples, and mostpreferably at least 99% of the samples.

The present invention further provides an antispirochete antimicrobialcomposition comprising a tetracycline antibiotic and a quinaloneantibiotic.

The present invention also provides a method of treating a subjecthaving symptoms of multiple sclerosis comprising: providing a subjecthaving symptoms of multiple sclerosis; and treating the subject with anyof the above antimicrobial compositions.

The present invention further provides a method of treating a subjecthaving symptoms of rheumatoid arthritis comprising: providing a subjecthaving symptoms of rheumatoid arthritis; and treating the subject withany of the above antimicrobial compositions.

The present invention also provides a method of treating a subjectsuspected of having a spirochetal infection comprising treating thesubject with any of the above antimicrobial compositions.

The present invention further provides a method of treating a subjecthaving symptoms of multiple sclerosis comprising: providing a subjectwith symptoms of multiple sclerosis; and a composition comprising atetracycline antibiotic and a quinalone antibiotic; and administeringthe composition to the subject. In some embodiments, the tetracyclineantibiotic is minocycline and the quinalone antibiotic is ciprofloxacin.

DESCRIPTION OF THE FIGURES

FIG. 1 presents a sequence fragment of B. burdorferi surface protein Afrom a human patient sample.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for culturingspirochetes and treating spirochetal diseases. For example, the presentinvention provides serum-free media for culturing spirochete bacteria invitro. The present invention further provides methods for identifyingspirochete susceptibilities to antimicrobials (e.g., antibiotics) andantimicrobial compositions and cocktails. The present invention alsoprovides methods for treating subjects suspected of having a spirocheteinfection.

For example, the present invention provides methods and media forculturing spirochetes. In some embodiments of the present invention, themedia is a defined, serum-free media for culturing any and allspirochete species in vitro. The methods and media of the presentinvention represent the first reliable serum-free media for culturingspirochetes and provide the only in vitro method available for culturingTreponemes. Thus, the present invention provides powerful new systemsfor culturing spirochetes, allowing spirochete detection andcharacterization.

The present invention also provides methods and compositions forspirochete detection. For example, in some embodiments of the presentinvention, samples suspected of containing at least one spirochetespecies or subspecies are cultured in the media of the present inventionto produce a sufficient number of organisms to permit their readydetection. It is contemplated that samples from any source will be usedin the implementation of the present invention. For example,environmental samples (e.g., water, soil, etc.), as well as clinical andveterinary samples will be used in conjunction with the presentinvention. Indeed, it is contemplated that spirochetal transmission willalso be assessed using the present invention (e.g., the use of cordblood to detect maternal transmission of organisms to the fetus inutero).

Importantly, the methods and compositions of the present inventionfacilitate determinations regarding the role of spirochetes inparticular disease states or patient symptoms, including diseases notcurrently associated with a spirochete etiology. For example, thepresent invention demonstrates an association between spirochetalinfection and the presentation of patient symptoms and diagnosis ofdiseases such as multiple sclerosis, arthritis, as well as otherdiseases (e.g., neurological and autoimmune diseases).

The present invention also provides methods and compositions forconducting antimicrobial susceptibilities, including methods andcompositions suitable for use in testing spirochetes for which nomethods are currently available. In one embodiment of the presentinvention, the media of the present invention is used to culture asufficient amount of spirochete organism to allow treatment with one ormore antimicrobials (e.g., in an antimicrobial cocktail). Thus, thesemethods identify antimicrobials that may be used to decontaminate asample (e.g., a water sample) or to treat subjects infected with aspirochete. These methods of the present invention also allow for theidentification of antimicrobials or antimicrobial cocktails specific toa certain patient or class of patients and allow for the identificationof antimicrobials or antimicrobial cocktails that are statistically mostlikely to successfully treat diseases due to any spirochete.

Thus, in some embodiments, the present invention provides methods andcompositions for treating patients infected with spirochetes and/orpatients suspected of experiencing spirochete infection (e.g., patientsexhibiting symptoms or disease states associated with a spirochetalinfection). In some embodiments of the present invention, a sample istaken from a patient and cultured. In the most preferred embodiments, atleast one medium of the present invention is inoculated with the patientsample and the spirochetes allowed to grow in the culture. The expandedspirochete culture is tested in an antibiotic susceptibility test panelto identify antimicrobial agents that effectively target the patient'sspirochetes. One or more of the suitable antimicrobial agents isadministered to treat the patient and eliminate the disease and/orinfection. Experiments conducted during the development of the presentinvention have demonstrated great success in human subjects. Forexample, patients exhibiting symptoms of multiple sclerosis and Lymedisease were individually tested for spirochete susceptibility andtreated with an antibiotic cocktail specifically designed for theirinfection. Undesired symptoms of the diseases were successfullyameliorated. However, in other embodiments, an antimicrobial orantimicrobial cocktail shown to be effective by the methods of thepresent invention is directly administered to the patient withoutconducting a susceptibility analysis for the specific patient.

The methods and compositions of the present invention thus providedramatic new systems for culturing spirochetes, identifying spirocheteinfections or contamination, identifying disease states associated withspirochete infection, and effectively treating patients having aspirochete infection. For example, the present invention hasdemonstrated a link between spirochete infection and patients diagnosedwith diseases such as multiple sclerosis (a disease that affects overone million people and has no known cure). The methods and compositionsof the present invention have been used to dramatically improve theconditions of such patients.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “microbiological media” and “culture media,”and “media” refer to any substrate for the maintenance, growth, andreproduction of microorganisms. “Media” may be used in reference tosolid plated media which support the growth of microorganisms. Alsoincluded within this definition are semi-solid and liquid microbialgrowth systems including those incorporating living host organisms, aswell as any type of media.

As used herein, the term “host” refers to any animal including insectsand warm blooded mammals. Warm blooded mammals include, but not are notlimited to, humans, non-human primates, rodents, and the like, which isto be the recipient of a particular treatment. Typically, the terms“host” and “patient” are used interchangeably herein in reference to ahuman or mammalian subject.

As used herein, the term “non-human animals” refers to all non-humananimals. Such non-human animals include, but are not limited to,vertebrates such as rodents, non-human primates, ovines, bovines,ruminants, lagomorphs, porcines, caprines, equines, canines, felines,ayes, etc.

As used herein, the term “mycoplasma” refers to members of the familyMycoplasmataceae—highly pleomorphic, gram-negative, aerobic tofacultatively anaerobic microorganisms differing from bacteria in thatthey lack a cell wall and are bounded by a triple-layered membrane.Mycoplasma are the smallest known free-living organisms, and include thepleuropneumonia-like organisms (PPLO), and are separated into species onthe basis of source, glucose fermentation, and the growth on agar media.

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On the one hand they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, and saliva, as well as solidtissue. These terms also refers to swabs and other sampling deviceswhich are commonly used to obtain samples for culture of microorganisms.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, disposable and non-disposable items. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for the growth,detection, and antimicrobial testing of spirochetes and providescompositions and methods for the treatment of samples and patientsinfected with spirochetes. Certain preferred embodiments of the presentinvention are described in detail below. The present invention is notlimited to these particular described embodiments. The description isprovided in the following section: I) Culture media; II) Spirochetedetection; III) Antimicrobial susceptibility testing; IV) Antimicrobialsand antimicrobial cocktails; V) Treatment of infected samples andpatients; and VI. Therapeutic Preparations And Combinations.

I) Culture Media

The present invention provides media for culturing spirochete organisms.The media of the present invention is the only media available that willculture all spirochete species in vitro, including the Treponemes. Inpreferred embodiments of the present invention, the media is serum-free.Standard currently available media for culturing certain spirochetespecies contain serum and do not efficiently and reliably grow theorganisms. They are particularly inefficient and unreliable at growingspecies and strains different from those they were optimized for.Because the variability inherent in the variety of spirochete organisms,this limitation is substantial. For example, experiments conductedduring the development of the present invention included testing ofvarious formulae of media of the present invention against BSK-H mediafor the growth of Borrelia species using serum samples obtained frompatients diagnosed with Lyme disease. BSK-H, a media optimized for aspecific strain of Borrelia, failed to culture and detect spirochete inapproximately 50% of the samples detected using media of the presentinvention.

The media of the presently claimed invention contain the nutrientsneeded for the metabolism and growth of spirochetes, including but notlimited to carbohydrate, salt, nitrogen, metallic cation, growthfactors, neural component, amino acid, and lipid sources. In preferredembodiments, the media is serum-free media. One skilled in the art willappreciate that various analogues, conjugates, complexes and the likemay be substituted for the ingredients recited herein without departingfrom the spirit of the invention.

In preferred embodiments the source of carbohydrates includes glucose,sucrose, fructose, lactose, and mannose. While other carbohydrates mayalso be included, the above carbohydrates are sufficient for the growthof spirochetes using the media of the present invention. In preferredembodiments the carbohydrates are provided as (+/−10%): 1.25 g/Lglucose, 1 g/L sucrose, 1.3 g/L fructose, 1.25 g/L lactose, and 1.2 g/Lmannose.

In preferred embodiments the source of salts includes sodium chloride,potassium chloride, lithium chloride, calcium chloride (2H₂O), magnesiumsulfate (anhydrous), potassium phosphate (monobasic anhydrous), andsodium phosphate (dibasic anhydrous). While other salts may also beincluded, the above salts are sufficient for the growth of spirochetesusing the media of the present invention. In preferred embodiments thesalts are provided as (+/−10%): 9 g/L sodium chloride, 1.8 g/L potassiumchloride, 0.5 g/L lithium chloride, 0.185 g/L calcium chloride (2H₂O),0.100 g/L magnesium sulfate (anhydrous), 0.060 g/L potassium phosphate(monobasic anhydrous), and 0.05 g/L sodium phosphate (dibasicanhydrous).

In preferred embodiments, the source of nitrogen includes peptone andneopeptone. While other sources of nitrogen may also be included, theabove sources are sufficient for the growth media of the presentinvention. In preferred embodiments, the sources of nitrogen areprovided as (+/−10%): 2.22 g/L peptone and 1.998 g/L neopeptone.

In preferred embodiments, the media of the present invention includemetallic cations; in particularly preferred embodiments, the metalliccations are manganese and magnesium. While other metallic cations mayalso be included, the above metallic cations are sufficient for thegrowth of spirochetes using the media of the present invention. Inpreferred embodiments, the metallic cations are provided as (+/−10%):0.052 g/L manganese and 0.044 g/L magnesium.

In preferred embodiments, the source of growth factor includes insulincomponents. While other growth factors may also be included, insulincomponents are sufficient for the growth of spirochetes using the mediaof the present invention. In some embodiments, factors that stimulatethe insulin receptor and/or factors that initiate an insulin-like signaltransduction cascade may be employed. In preferred embodiments, theinsulin components are provided as 0.500 ml/L SITE+3 liquid mediasupplement and 0.250 ml/L N 1 medium supplement.

In preferred embodiments, the source of neural components include brainextract type 1, brain extract type 6, and sphingomyelin (Sigma). Whileother sources of neural components may also be included, the abovesources are sufficient for the growth media of the present invention. Inpreferred embodiments, the sources of neural components are provided as:0.025 g/L brain extract type 1, 0.300 g/L brain extract type 6, and0.053 g/L sphingomyelin.

In preferred embodiments, the source of amino acids includes RPMI 1604(50×), MEM essential amino acids, and MEM non-essential amino acids(Sigma). While other sources of amino acids may be included orsubstituted, the above sources are sufficient for the growth media ofthe present invention. In preferred embodiments, the sources of aminoacids are provided as: 1 ml/L RPMI 1604 (50×), 10 g/L MEM essentialamino acids, and 10 ml/L MEM non-essential amino acids.

In preferred embodiments, the source of lipids includes concentratedlipid solution (1000×) (Sigma). While other sources of lipids may beincluded or substituted, the above source is sufficient for the growthmedia of the present invention. In preferred embodiments, theconcentrated lipid solution (1000×) is provided at 0.100 ml/L.

In preferred embodiments, the media further comprises insect mediacomponents. For example, in certain embodiments of the present inventionthe media comprises pluronic F-68 (10%) solution and Vanderzandt'ssupplement. In preferred embodiments, the pluronic F-68 (10%) solutionis provided at 2 ml/L and the Vanderzandt's supplement is provided at0.530 g/L (+/−10%).

In preferred embodiments, the media further comprises propellantcomponents. For example, in certain embodiments of the present inventionthe media comprises L-methionine, sodium pyruvate, and L-glutamine. Inpreferred embodiments, the L-methionine is provided at 5 ml/L, thesodium pyruvate is provided at 22.64 g/L, and the L-glutamine isprovided at 4.6 g/L (+/−10%).

In preferred embodiments, the media further comprises cell wallcomponents. For example, in certain embodiments of the present inventionthe media comprises N-acetyl glucosamine. In preferred embodiments, theN-acetyl glucosamine is provided at 5 g/L.

In preferred embodiments, the media further comprises endocrinecomponents.

For example, in certain embodiments of the present invention the mediacomprises thyroxine, thyroid powder, and estradiol. In preferredembodiments, the thyroxine is provided at 0.12 g/L, thyroid powder at0.22 g/L, and estradiol 0.008 g/L.

In preferred embodiments, the media further comprises additionalcomponents. For example, in certain embodiments, the present inventionprovides glycine, ferrous sulfate, muscle extract powder, myelin basicprotein lactalbumin hydrolysate, granulated yeast, sodium bicarbonate,Hepes (all from Sigma), and brain heart infusion (Difco). In preferredembodiments, the additional components are provided as: 1.6 g/L glycine,0.05 g/L ferrous sulfate, 0.51 g/L muscle extract powder, 0.001 g/Lmyelin basic protein, 10 g/L lactalbumin hydrolysate, 0.53 g/Lgranulated yeast, 0.05 g/L sodium bicarbonate, 5.25 g/L Hepes, and 0.66g/L brain heart infusion.

The media of the present invention has been shown to successfullyculture every type of spirochete tested, including various strains ofTreponemas, Borrelias, and Leptospires. Furthermore, significantpopulation growth of the organisms is provided by the media. Inaddition, any sample type may be used to inoculate the media. Forexample, sample such as serum, water, tissue, urine, cerebrospinalfluid, semen, amniotic fluid, fetal cord blood have been culturedsuccessfully. Other samples, including, but not limited to lesionexudates, synovial fluid, skin, etc., may be used to inoculate the mediaof the present invention. However, experiments conducted during thedevelopment of the present invention demonstrated that samples (bloodsamples) taken from sterile tubes containing a citrate preservative or aheparinized preservative yielded little to no growth. Thus, samplesplaced in such preservatives should be avoided. The media of the presentinvention has also been shown to successfully culture Babesia microtiand Ehrlichia species, tick-borne microorganisms associated withsignificant disease in humans and lower animals, and which may be foundin conjunction with spirochetes (i.e., co-infections).

The media of the present invention are suited for use as primary culturemedia as well as for maintenance of spirochete cultures. For example,spirochetes may be passaged multiple times using the media of thepresent invention. However, it was observed during the development ofthe present invention that, in some embodiments, after 6 to 7sub-passages are performed, the isolate is likely to be attenuated fromits original state, typical of bacteriologic cultures grown in vitro. Insome embodiments of the present invention, the media of the presentinvention finds use as a selective medium. For example GSI-I medium(Example 1) is a selective media as many non-spirochete species ofbacterial are not grown in the medium. The medium is specific foranaerobic or microaerophilic species. Typical of these are thespirochetes other tick-born infections.

In contrast to other media currently available, the media of the presentinvention may be incubated at room temperature. Indeed, cultures may bemaintained in the media of the present invention over a wide temperaturerange. For example, cultures grown at both room temperature and 37° C.have demonstrated good growth, although faster growth rates aretypically obtained with the higher temperature. For example, as comparedto room temperature, maintaining the incubation at 33-35° C. was shownto improve the initial growth phase of some organisms. Data also showedthat incubation at 33-35° C. resulted in an increase from 0-1organisms/field (phase microscopy) at time zero to 5-6 organisms/fieldwithin 3-4 days of incubation. Continued incubation at either theelevated or room temperature resulted in 10-12 organisms/field by 14days of incubation. Maintaining the culture at room temperaturethroughout the time course requires approximately 10 extra days to reach10-12 organisms/field.

Confirmation of growth of the desired organism, if desired, may beconducted using any number of methods. For example, successfulconfirmation has been achieved through immunofluorescence antibodystaining, visualization by light or phase contrast microscopy, PCRamplification of spirochete-specific markers (e.g., amplification usingprimers encompassing a portion of the Osp A gene of B. burgdorferi),sequencing, and visualization by electron microscopy.

II) Spirochete Detection

The present invention provides novel methods and compositions for thedetection of spirochetes. For example, the presence of spirochetes in asample may be achieved by inoculating the media of the present inventionwith the sample or an aliquot of the sample. The inoculated media isincubated until a sufficient amount of organism is generated to allowtheir detection or characterization using the chosen method (e.g.,immunofluorescence antibody staining, microscopy, PCR amplification ofspirochete-specific markers, sequencing, and the like). If the sample isto be used in susceptibility testing, as described below, additionalincubation may be required to achieve sufficient population ofspirochete for testing.

In certain embodiment of the present invention spirochetes such asLeptospires are detected in water samples or other environmental orindustrial samples suspected of containing a spirochete. For example,the media of the present invention is inoculated with a portion of thewater sample and incubated until detectable levels of spirochetes areobtained. Spirochetes are detected using any of the methods describedherein, or any other suitable methods. Samples containing spirochetesmay undergo further characterization such as identification ofparticular stains of spirochete or antimicrobial susceptibility testing(described below). Samples containing spirochetes may be treated with anappropriate antimicrobial agent to remove or reduce the spirochetecontent, or the sample may simply be avoided (e.g., human or animalcontact with the water or other contaminated substance prevented oravoided).

In other embodiments of the present invention tissues or fluid samplesfrom a subject are tested for the presence of spirochetes. Subjectsinclude both humans, non-human mammals (e.g., livestock), and otheranimals. The media of the present invention is inoculated with thesample and spirochetes are cultured and detected as described above andin the Examples. Samples infected by spirochetes may undergo furthercharacterization such as identification of particular stains ofspirochete or antimicrobial susceptibility testing (described below).Subjects infected with spirochetes may be treated with an appropriateantimicrobial agent to remove or reduce the spirochete load. Detectionof spirochetes in human subject may also be used to detect, monitor,and/or prevent mother-to-child transmission of spirochete infection inutero. As shown in Example 17, the present invention describes suchtransmissions.

The detection methods of the present invention may also be used toidentify new spirochetes (e.g., new strains and species of spirochetesand genetic variants of new or known spirochetes) and to identify theassociation of particular spirochetes with certain samples (e.g., watersamples) or conditions (e.g., disease states). For example, use of thepresent invention has demonstrated an association between certainspirochete strains and human disease states such as multiple sclerosisand rheumatoid arthritis. Whether the spirochete is the direct and solecausative agent of such disease states or whether it acts to induce orexacerbates the disease state is unknown. However, such knowledge is notrequired for the successful practice of the present invention of thepresent invention, and the present invention is not limited to anyparticular causative mechanism of action. Rather, the present inventiondemonstrates and provides means to demonstrate that many patientsdiagnosed with such diseases, and suffering from symptoms associatedwith such diseases, are infected with spirochetes. Treatment withantimicrobial agents specific for the spirochete, and in someembodiments specific for a particular patient, have been shown toameliorate the symptoms of the disease and dramatically improve patientcondition. Thus, detection of spirochete infection in certain classes ofpatients provides novel methods and therapies for treating suchpatients.

The case of multiple sclerosis provides a compelling example.Experiments conducted during the development of the present inventioninvolved testing of 146 patient samples from patients with a history ofmultiple sclerosis. Using media (GSI-1 media described in Example 1) andmethods of the present invention, all 146 of the patients testedpositive for a Leptospiral infection. Three of the 146 patients alsotested positive for Borrelia burgdorferi infection. Interestingly, thesethree patients exhibited particularly severe symptoms of multiplesclerosis. Thus, the use of the present invention has resulted in theidentification of a new target and provides new approaches for treatingpeople exhibiting symptoms of multiple sclerosis. Importantly fortreatment of affected individuals, current treatment regimes formultiple sclerosis do not employ any anti-bacterial agents, let aloneanti-spirochetal agents or anti-spirochetal agents customized for aparticular patient. For example, current research focuses on the use ofintravenous immunoglobulins, plasma exchange, antiviral medications, TCRand T-cell vaccines, vitamin D therapy, bone marrow transplantation, andbee venom, among others. Most current treatments simply attempt toalleviate symptoms of the disease. Whether or not multiple sclerosis isa class of different diseases or whether many people are misdiagnosed,it is clear that a great majority of diagnosed patients are infectedwith spirochete that is detected and treated by the compositions andmethods of the present invention.

The present invention has also found an association between rheumatoidarthritis and infection by a Leptospira. In view of the neurological andautoimmune conditions (See also, Lyme disease) associated withspirochete infection, it is contemplated that a variety of autoimmuneand neurological diseases and patients exhibiting symptoms of autoimmuneand neurological disease are infected with spirochetes, which may bedetected and treated using the compositions and methods of the presentinvention. It is contemplated that diseases and symptoms of diseasesincluding, but not limited to, multiple sclerosis, rheumatoid arthritis,osteoarthritis, lupus erythrematosis (both system and discoid), coronaryartery disease, amyotrophic lateral sclerosis, alzheimer's disease,chronic fatigue syndrome, ankylosing spondylitis, diabetes,hypoglycemia, depression, sleep disorders, Grave's disease, Hashimoto'sdisease, and Lyme disease (including gestational Lyme, neuroborreliosis,and pediatric lyme) may have a spirochetal component.

Detection methods of the present invention have successfully shown thatspirochetes associated with particular disease states have consistentmorphologies. For example, the leptospires found in patients diagnosedwith multiple sclerosis have similar morphologies that differ fromleptospires and spirochetes found in other types of patients (See,Example 11). Thus, the present invention provides novel methods foridentifying particular disease states based on the morphology of thespirochete isolated from a patient. In some embodiments of the presentinvention, spirochetes from the patient are examined by phase contrastor electron microscopy to determine morphology. In other embodiments ofthe present invention, genetic signatures of spirochetes are examinedand used to diagnose the disease state. For example, spirochete nucleicacid may be sequenced or used in a hybridization assays withstrain-specific probes to visualize and analyze the morphology of theisolated organism.

Diagnosis of disease states may also be accomplished through theco-detection of other agents with a spirochete. For example, experimentsconducted during the development of the present invention havedetermined that rheumatoid arthritis patients show the presence of amycoplasma in the vicinity of or directly associated with a spirochete.Thus, the co-detection of spirochete and mycoplasma provides aindication of rheumatoid arthritis.

With respect to water samples and other environmental samples, thepresent invention provides efficient methods for filtering the samplesto extract spirochetes. For example, in one embodiment of the presentinvention antibodies or other binding agents specific for a spirochetedetected in a sample are fixed to a solid support (e.g., filters) andused to eliminate spirochetes from the sample (e.g., water flows in awater treatment facility).

III) Antimicrobial Susceptibility Testing

The present invention provides methods for conducting antimicrobialsusceptibility testing of spirochetes. Samples containing a spirocheteare cultured to obtain a sufficient amount of bacteria to allowsusceptibility testing. Samples are then treated with one or moreantimicrobials and growth and survival of the spirochete is examined.Such methods allow for the detection of anitmicrobials that are lethalto particular spirochetes and/or classes of spirochetes.

Due to the difficulty of cultivating spirochetes among other problems(e.g., fastidious growth requirements and microaerophilic environment),prior to the present invention, easy to perform antimicrobialsusceptibility tests suitable for use with all spirochetes wereunavailable. However, using the methods and media of the presentinvention, sensitivities can be reliably and easily performed. Although,in some cases, susceptibility tests are directly performed on culturedsamples (i.e., cultured using the media of the present invention),several technical problems had to be surmounted in order to allow thesensitivities of the present invention to work generally. For example, areliable media was required. Additionally, the total number ofspirochetes needed to be expanded and maximized in order to provide asufficient number of organisms for testing. In some embodiments of thepresent invention, this was accomplished by centrifugation anddisruption of the spirochetes. For example, in one embodiment of thepresent invention, spirochetes are disrupted by treatment with 10% SDSsolution at 14,000 g so that the maximum number of epitopic sites areavailable.

The next technical hurdle was to develop methods and media to maximizethe concentration of spirochetes. In one embodiment of the presentinvention this was accomplished by using an alternative growth mediumcontaining an increased lactalbumin hydrolysate and brain heart infusionconcentrations. In a preferred embodiment, the medium comprises one partGSI-1 medium, one part of 6.5% lactalbumin hyrdolysate, one part 2%brain heart infusion, and one part 10% pluronic fluid. The medium wasinoculated with isolate and allowed to incubate in a closed sterilemicrocentrifuge tube until a distinctive pellet was observed.

For susceptibility testing, the organisms harvested could be assayedusing any suitable susceptibility assay (See, Example 7). In someembodiments of the present invention, MICROSCAN antibioticsusceptibility plates were inoculated and incubated. Followingincubations, plates were scanned for growth and respective sensitivityreaction. Each MICROSCAN plate had a positive control (growth) well anda corresponding negative control well. For the results to be consideredreliable, the no growth of organisms was accepted in the negativecontrol well, while the positive control well had to have sufficientgrowth to be read. In some embodiments of the present invention, toconfirm the presence or absence of growing spirochetes in the growthwell, aliquots from the growth well were reinoculated with fresh growthmedia and after an incubation time (e.g., 7-10 days) the presence of theorganisms was confirmed (e.g., by phase contrast microscopy or directfluorescent antibody staining).

In some embodiments of the present invention susceptibility assays areconducted on samples suspected of containing a spirochete to determinespecific antimicrobial agents capable of killing or inhibiting thespecific spirochete present in the sample. Because of the variability inspirochete species and strains, such methods are desired in order toselect effective treatments. For example, antimicrobial susceptibilityassays provide a determination of the appropriate antimicrobialtreatment to be used originally in an infected patient. In addition,these methods provide means to monitor the efficacy of the treatment.Such determinations are useful to detect and counter any resistance thatdevelops in the patient's culture. For example, for long term therapies(e.g., treatment over a period of years), repeated susceptibilitytesting is desired to optimize ongoing treatments as particularantimicrobials become less effective.

In other embodiments of the present invention susceptibility assays areconducted to determine specific antimicrobials, classes ofantimicrobials, and/or antimicrobial cocktails that may be usedgenerally (e.g., against a certain genus, species, or strain ofspirochete). For example, in some embodiments of the present invention,samples from a suitable number of patients with a particular disease orspirochetal infection are obtained and assayed. Antimicrobials thateffectively target a high percentage of the patients are identified.These high percentage antimicrobials are then used to treat patientshaving the particular disease or spirochetal infection. In someembodiments of the present invention, two or more of the antimicrobialsthat collectively treat a high percentage of patients are identified. Insuch embodiments, individual antimicrobials may only affect a portion ofthe population, but the collective effect of the combination providesthe desired certainty. In preferred embodiments of the presentinvention, 80% or more of the spirochete samples tested are susceptibleto the antimicrobial or combination. In particularly preferredembodiments, 90%, 95%, 98%, 99%, or 99.5% of spirochete samples aresusceptible. In preferred embodiments, the total number ofantimicrobials used to achieve the desired susceptibility rate isminimized and in particularly preferred embodiments the total number isthree or less.

IV) Antimicrobials and Antimicrobial Cocktails

The present invention provides antimicrobials and antimicrobialcocktails that are useful in the treatment of spirochetal disease andinfection. As described above, the identity of antimicrobials andantimicrobial cocktails that are effective for a particular disease orspirochete may be determined using the susceptibility assays of thepresent invention. The present invention contemplates that anyantimicrobial may find use with the present invention. Any agent thatcan kill, inhibit, or otherwise attenuate the function of spirochete maybe used, as well as any agent contemplated to have such activities.Antimicrobial agents include, but are not limited to, natural andsynthetic antibiotics, antibodies, inhibitory proteins, antisensenucleic acids, membrane disruptive agents and the like, used alone or incombination.

Indeed, any type of antibiotic may be used including, but not limitedto, anti-bacterial agents, anti-viral agents, anti-fungal agents, andthe like. In preferred embodiments the antimicrobial of the presentinvention is an antibiotic. The present invention is not limited to anyparticular type of antibiotic. Antibiotics that find use in the presentinvention include, but are not limited to, Acyclovir (Zovirax),Amantadine (Symmetrel), Amikacin (generic), Gentamicin, Tobramycin,Amoxicillin, Amoxicillin/Clavulanate (Augmentin), Amphotericin B(Fungizone), Ampicillin, Ampicillin/sulbactam (Unasyn), Atovaquone(Mepron), Azithromycin (Zithromax), Cefazolin, Cefepime (Maxipime),Cefotaxime (Claforan), Cefotetan (Cefotan), Cefpodoxime (Vantin),Ceftazidime, Ceftizoxime (Cefizox), Ceftriaxone (Rocephin), Cefuroxime(Zinacef), Cephalexin, Chloramphenicol, Clotrimazole (Mycelex),Ciprofloxacin (Cipro), Clarithromycin (Biaxin), Clindamycin (Cleocin),Dicloxacillin, Doxycycline, Erythromycin (including estolate,ethylsuccinate, gluceptate, lactobionate, and stearate), Famciclovir(Famvir), Fluconazole (Diflucan), Foscarnet (Foscavir), Ganciclovir(Cytovene), Imipenem/Cilastatin (Primaxin), Isoniazid, Itraconazole(Sporanox), Ketoconazole, Metronidazole (Flagyl), Nafcillin,Nitrofurantoin, Nystatin, Penicillin (including G benzathine, Gpotassium, G procaine, V potassium), Pentamidine,Piperacillin/Tazobactam (Zosyn), Rifampin (Rifadin),Ticarcillin/Clavulanate, Trimethoprim, Trimethoprim Sulfate,Valacyclovir (Valtrex), Vancomycin, Aztreonam, Levofloxacin (Levaquin),Meropenem, Tobramycin, Cephalothin (Tazidime), Mezlocillin, Nalidixicacid, Netilmicin, Minocycline, Ofloxacin, Norfloxacin, Sulfamethoxazole,Tetracycline, Neomycin, Streptomycin, Cephalosporin, Ticarcillin,carbenicillin, cloxacillin, Cefoxitin, ceforanide, teicoplanin,ristocetin, viomycin, capreomycin, bacitracin, gramicidin, gramicidin S,tyrocidine, Tachyplesin, kanamycin, methicillin, oxacillin, azocillin,bacampicillin, carbenicillin indanyl, cephapirin, cefaxolin, cephradine,cefradoxil, cefamandole, cefaclor, cefuromime axetil, cefonicid,cefoperazone, demeclocytetracycline, methacycline, oxytetracycline,spectinomycin, ethambutol, aminosalicylic acid, pyrazinamide,ethionamide, cycloserine, dapsone, sulfoxone sodium, clofazimine,sulfanilamide, sulfacetamide, sulfadiazine, sulfixoxazole, cinoxacin,methenamine, and phenazopyridine. The present invention contemplate thatany other antibiotic now known or discovered in the future may be usedin the methods and compositions of the present invention (e.g., may beused in susceptibility assay to determine ability to treat spirochetalinfections).

Experiments conducted during the development of the present inventionidentified a variety of antibiotic and antibiotic cocktails that findparticular use in treating spirochetal infections. For example, theresults of several hundred patient samples having Borrelia andLeptospira infections were tested using the susceptibility methods ofthe present invention. The results are present in Example 8. As is clearfrom this data, there are a variety of antibiotics and antibioticcombinations that achieve very high success rates. However, individualpatients may not respond to any particular high percentage antibiotic.For example, as shown in Example 7, a susceptibility profile from aspecific human patient showed resistance to augmentin, an antibioticwith over 80% effectiveness in the population, but was sensitive toaztreonam, an antibiotic with less than 20% effectiveness in thepopulation. Thus, in some embodiments of the present invention, asusceptibility assay is performed on cultures from individuals todetermine the appropriate antibiotic treatment or, in the alternative, aantibiotic cocktail is used. Preferably, the cocktail contains only twoor three high percentage antibiotics to minimize unnecessary overuse ofantibiotics, selection for broad spectrum resistance, and unwantedside-effects, while maximizing likelihood of success (i.e., as opposedto using a shot-gun approach).

Experiments conducted during the development of the present inventionhave identified certain specific antibiotic cocktails that areparticularly efficacious. In preferred embodiments of the presentinvention the antibiotic cocktail comprises a tetracycline antibiotic(e.g., demeclocytetracycline, doxycycline, methacycline, minocycline,and oxytetracycline). In particularly preferred embodiments, thetetracycline antibiotic is doxycycline or minocycline. In preferredembodiment the cocktail further comprises a quinalone (e.g., nalidixicacid, cinoxacin, norfloxacin, gentamicin, azithromycin, clarithromycin,levofloxacin, ofloxacin, and ciprofloxacin, preferably ciprofloxacin), amacrolide, and/or a beta-lactam (e.g., amoxicillin and augmentin). Inother embodiments, the cocktail comprises a tetracycline and aaminoglycoside (e.g., amikacin, gentamicin C, kanamycin A, neomycin B,netilmicin, streptomycin, and tobramycin) and/or a cephalosporin (e.g.,cephapirin, cefaxolin, cephalexin, cephradine, cefadroxil, cefamandole,cefoxitin, cefaclor, cefuroxime, cefuroxime axetil, cefonicid,cefotetan, ceforanide, cefotaxime, ceftizoxime, ceftriaxone,cefoperazone, and ceftazidime). Other combination that find use incertain embodiments of the present invention includeampicillin/solbactam, ticarcillin/clavulanate, piperacillin/tazobactam,amoxicillin/clavulanate, and trimethoprim/sulfamethoxazole.

V) Treatment of Infected Samples and Subjects

The present invention provides methods and composition for the treatmentof infected materials (e.g., water sources and food sources) andsubjects (e.g., humans, as well as, other animals) infected with aspirochete. As described above, the present invention provides methodsand compositions for the detection of spirochetal infections and methodsand compositions for the identification of antimicrobials effective atreducing or eliminating the infections. Thus, the present inventionprovides all of the steps necessary for diagnosing spirochete infectionand provides appropriate treatments. In some embodiments of the presentinvention, detection is not required prior to treatment. For example,samples suspected of containing a spirochete or subjects having symptomsconsistent with a spirochete infection (e.g., neurological andautoimmune symptoms) or having a disease diagnosis associated with aspirochetal infection are directly treated with antimicrobials (e.g.,antimicrobial cocktails developed by the methods of the presentinvention to be statistically likely to treat a spirochete infection)without first detecting the presence of a spirochete (i.e., the patientsare treated empirically using knowledge gained by the practice of thepresent invention).

The antimicrobial treatments of the present invention can beadministered by any number of routes and in any number of forms. Forexample, the treatments may be administered by routes including, but notlimited to, orally, topically, rectally, vaginally, by pulmonary route(e.g., by use of an aerosol), or parenterally, including, but notlimited to, intramuscularly, subcutaneously, intraperitoneally,intracranially, intrathecally, or intravenously. The compositions can beadministered alone, or can be combined with apharmaceutically-acceptable carrier, adjuvant, or excipient according tostandard pharmaceutical practice.

In embodiments where the subject is treated for neurological symptoms orwhere the spirochete is suspected to be in neural tissues, thetreatments of the present invention may be modified or utilized inmethods that promote activity across the blood-brain barrier. Theblood-brain barrier is a capillary barrier comprising a continuous layerof tightly bound endothelial cells. These cells permit a low degree oftransendothelial transport, and exclude molecules in the blood fromentering the brain on the basis of molecular weight and lipidsolubility.

In one embodiment of the present invention, the treatment compositionsof the present invention are conjugated to carrier molecules that assistthe compounds of in traversing of the brain-blood barrier (See e.g.,U.S. Pat. Nos. 4,540,564, 4,771,059, 4,824,850, and 5,296,483, all ofwhich are herein incorporated by reference in their entireties). Inother embodiments, the treatment compositions of the present inventionare administered to a subject that has been subjected to one or acombination of techniques that increase the permeability of patient'sblood-brain barrier to therapeutic compounds. An example of such amethod is described in U.S. Pat. No. 5,752,515, herein incorporated byreference in its entirety. In still further embodiments, compounds knownto increase the permeability of the blood-brain barrier areco-administered with the treatment compositions of the presentinvention. Example of such compounds are described in U.S. Pat. Nos.5,112,596, 5,154,924, 5,268,164, 5,506,206, and 5,686,416, all of whichare herein incorporated by reference in their entireties. In yet otherembodiments the treatment compositions of the present invention aredelivered to a patient at predetermined sites directly into the brain orneural tissue. For example, U.S. Pat. No. 5,792,110 (incorporated hereinby reference in its entirety) teaches a delivery system for therapeuticagents that includes a guide cannula for penetrating a selected site ina subject to a predetermined depth and a delivery cannula for deliveringthe therapeutic agent to the subject. The ability of certain deliverysystems to provide efficacious delivery of particular treatmentcompositions can be tested using artificial in vitro blood-brain barriermodels (See e.g., U.S. Pat. No. 5,260,210, herein incorporated byreference in its entirety).

VI. Therapeutic Preparations and Combinations

In some embodiments, the present invention provides therapeuticcompositions of antimicrobials or antimicrobial cocktails (as describedin Section IV), and pharmaceutical agents typically used to treatdiseases such as Lyme disease, rheumatoid arthritis, leptospirosis, etc(e.g., leflunomide, etanercept, and infliximab for rhematoid arthritis).This combination therapy may further be combined with molecules used toassist traversal of the blood brain barrier. It is not intended that thepresent invention be limited by the particular nature of the therapeuticcomposition. For example, such compositions can be provided togetherwith physiologically tolerable liquids, gels, solid carriers, diluents,adjuvants and excipients (and combinations thereof). Suitablepharmaceutical agents that may be combined with the antimicrobials ofthe present invention include, but are not limited to salicylate,steroids, immunosuppressants, or antibodies.

The therapeutic compositions of the present invention can beadministered to non-human animals (e.g., for veterinary use), such aswith domestic animals (e.g., livestock and companion animals), wildanimals, and non-human primates, and clinical use in humans in a mannersimilar to other therapeutic agents. In general, the dosage required fortherapeutic efficacy varies according to the type of use and mode ofadministration, as well as the particularized requirements of individualhosts. The attending medical professional is capable of determining thetherapeutically effective dosage based on the characteristics of thesubject.

Therapeutic compositions may contain such normally employed additives asbinders, fillers, carriers, preservatives, stabilizing agents,emulsifiers, buffers and excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, magnesium carbonate, and the like. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70%.

The therapeutic compositions of the present invention can also be mixedwith diluents or excipients which are compatible and physiologicallytolerable. Suitable diluents and excipients are, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired, the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents.

In some embodiments, the therapeutic compositions of the presentinvention are prepared either as liquid solutions or suspensions, assprays, or in solid forms. Oral formulations usually include suchnormally employed additives such as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%. One example of an oral composition useful fordelivering the therapeutic compositions of the present invention isdescribed in U.S. Pat. No. 5,643,602, incorporated herein by referencein its entirety.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, transdermal patches, and suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides. One exampleof a topical delivery method is described in U.S. Pat. No. 5,834,016,incorporated herein by reference in its entirety. Other liposomaldelivery methods may also be employed. In certain embodiments, thetherapeutic compositions are administered via a transdermal patch (Seee.g., U.S. Pat. Nos. 4,638,043, 5,830,505, and 5,876,746, all of whichare incorporated herein by reference in their entireties).

In other preferred embodiments, enteric formulations are employed. Thecovering may comprise an enteric coating or a capsule. The terms“enteric coating” or “enteric film” are used interchangeably and referto a material or compound that is resistant to acid pH (i.e., anacid-resistant compound), such as that found in the stomach. An entericcoating when applied to a solid inhibits the dissolution of the solid inthe stomach.

Standard techniques may be employed for the encapsulation of solidcompositions. These techniques include microencapsulation of a solidcomposition wherein an enteric coating is applied to the solidcomposition. The coated material may be delivered orally to a subject bysuspending the microencapsulated particles in pharmaceutical suspensionsolutions. The capsule preferably has the characteristic of beingresistant to dissolution in the stomach and being capable of dissolvingin the intestines. Numerous suitable capsule formulations are known; inaddition standard techniques are available for the filling of capsulesincluding the use of inert filler materials to provide sufficient bulkof the filling of a capsule with a therapeutic composition in a solidform. In addition to the use of encapsulated compositions, theantimicrobial therapeutic compositions of the present invention may bedelivered orally in tablet or pill form. The therapeutic compositions ofthe present invention may be combined with inert materials to providesufficient bulk for the pressing of the tablet or pill. Once formed, thetablet or pill may then be coated with an enteric film to preventdissolution in the stomach and to enhance dissolution in the intestines.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); ° C. (degrees Centigrade); and Sigma (Sigma Chemical Co.,St. Louis, Mo.).

Example 1 Preparation of GSI-1 Spirochete Culture Medium

This Example describes the preparation of a one liter sample of theGSI-1 type Spirochete culture medium of the present invention. Thevarious components used to make the media and their respectiveconcentrations are listed below in Table 1.

TABLE 1 Components Used to Make 1 Liter of GSI-I Medium CategoryComponent Amount Sugars (Sigma) Glucose 1.250 g/L Sucrose 1.000 g/LFructose 1.300 g/L Lactose 1.250 g/L Mannose 1.200 g/L Salts (Sigma)Sodium Chloride 9.000 g/L Potassium Chloride 1.800 g/L Lithium Chloride0.500 g/L Calcium Chloride-2H₂O 0.185 g/L Magnesium Sulfate 0.100 g/L(anhydrous) Potassium Phosphate 0.060 g/L monobasic (anhydrous) SodiumPhosphate Dibasic 0.050 g/L (anhydrous) Nitrogen (Difco) Peptone 2.220g/L Neopeptone 1.998 g/L Metallic Cations Manganese 0.052 g/L (Sigma)Magnesium 0.044 g/L Insulin SITE + 3 Liquid Media 0.500 ml/L ComponentsSupplement (Sigma) N 1 Medium Supplement 0.250 ml/L Amino Acids RPMI1640 (50 X) 1.000 ml/L (Sigma) MEM Essential Amino Acids 10.000 g/L MEMNon-Essential Amino 10.000 ml/L Acids Insect Media Pluronic F-68 (10%)Solution 2.000 ml/L Components Vanderzandt's Supplement 0.530 g/L(Sigma) Propellant L-Methionine 5.000 ml/L Components Sodium Pyruvate22.640 g/L (Sigma) L-Glutamine 4.600 g/L Neural Brain Extract Type 10.025 g/L Components Brain Extract Type 6 0.300 g/L (Sigma)Sphingomyelin 0.053 g/L Lipids (Sigma) Concentrated Lipid Solution 0.100ml/L (1000 X) Cell Wall N-Acetyl Glucosamine 5.000 g/L Component (Sigma)Endocrine Thyroxine 0.120 g/L Components Thyroid Powder 0.220 g/L(Sigma) Estradiol 0.008 g/L Other Components Glycine 1.600 g/L (Sigma)Ferrous Sulfate 0.050 g/L Muscle Extract Powder 0.510 g/L Myelin BasicProtein 0.001 g/L Lactalbumin Hydrolysate 10.00 g/L Granulated Yeast0.530 g/L Sodium Bicarbonate 0.050 g/L Brain Heart Infusion (Difco)0.660 g/L Hepes 5.250 g/L

The preparation of the GSI-1 media begins with approximately 400-500 mLof sterile water (for irrigation) poured into a sterile, gammairradiated plastic disposable receiver. A sterilized plastic magneticstirring bar is placed into the receiver and the assembly is placed on anon-heated magnetic stirring plate.

The solid components from Table 1 are added one at a time in any order,and allowed to mix for several hours at ambient room temperature. Afterthorough mixing has been achieved, the assembly and the mixed componentsare placed inside an operating Class II Laminar Flow Hood where theliquid components are added one at a time using aseptic tissue culturetechnique. The final volume of 1.00 Liter is achieved by addingadditional sterile water for irrigation. The combined mixture is allowedto stir for another hour, and then vacuum filtrated into a sterile,gamma irradiated plastic receiver through a 0.22 micron celluloseacetate membrane filter.

After the medium is prepared, it is tested for sterility. This is doneby inoculating several loopfuls of new media onto a fresh 5% sheep bloodagar plate and allowed to incubate overnight. If no colonies are presenton the plate the next day, it is ready for use as a culture medium forspirochete microorganisms. However, if colonies are present, the processis repeated. If two consecutive blood agar plate attempts are positivefor bacterial contamination, the medium is discarded. The sterile mediumis stored at 4° C. until use.

Example 2 Inoculating GSI-I Medium with a Sample

This examples describes inoculating GSI-1 medium with a sample in orderto culture spirochetes. To culture microorganisms on GSI-1,80% media isused for a 20% amount of inoculum (whether it be water or body fluidslike serum, urine, cerebrospinal fluid, synovial fluid, semen, amnioticfluid, or fetal cord blood). In this example, a sterile flask isassembled within a class II laminar flow hood along with therefrigerated GSI-1 culture medium prepared according to Example 1 and apatient serum sample in a capped test tube. The 80% volume of GSI-1media is aseptically transferred or pipetted into the sterile flask. Themedia is then recapped and placed back into the refrigerator forstorage. The test tube with the patient serum sample is then uncapped,and the 20% of serum is aseptically placed within the sterile flaskcontaining sterile GSI-1 media. The flask is then capped to ensure amicroaerophilic growth environment and incubated at room temperatureuntil microscopic examination is performed. At various time periods theinoculated culture sample is examined. Examination includes both visualidentification under a microscope and an antibody binding screen.

The GSI-1 culture media was also used to culture organisms present intissue samples. For example a cryopreserved biopsy material from a tickbite rash (i.e., erythema migrans) from a Lyme disease patient wascultured in the GSI-1 media. The tissue sample was approximately 2 by 4by 4 millimeters in volume. GSI-1 media was transferred to the sterileflasks. The cyropreserved erythema migrans biopsy sample was thentransferred into the sterile flask. The flask was then capped andincubated until microscopic examination was performed. At various timeperiods, the inoculated culture samples are examined. Examinationincludes methods such as visual identification under a microscope, anantibody binding screen, and PCR analysis.

Example 3 Use of GSI-1 Media to Screen for Spirochetes

This example describes screening of inoculated GSI-1 medium for thepresence of Treponema, Borrelia, and Leptospira. Five serum samplespreviously confirmed by assay for RPR (rapid plasma reagin) and MHA-TP(microhemagglutination assay for T. pallidum) were used in theseexperiments. Each of the five samples were inoculated in GSI-1 (e.g., inthe manner described in Example 2). At inoculation, less than oneorganism per field was observed by phase contrast microscopy in theseculture samples. The cultures were allowed to grow for 21 days at 37° C.Each of the cultures was then assayed using T. pallidum specificindirect and direct immunofluorescence antibody staining. Dilutions of1:10 and 1:20 were performed with the appropriate controls (e.g.,negative controls such as blanks and Leptospira antigen and positivecontrols such as a T. pallidum positive slide). After application, thereaction was allowed to incubate for 45 minutes at 37° C. prior tocounting. Examination by fluorescent microscopy demonstrated intact,motile spirochetes in each of the five culture samples. Examination byfluorescent microscopy also revealed that the number of organisms ineach culture sample had increased to approximately 10-14 organisms perfield, as compared to the 0-1 organisms present at inoculation. Thisexample makes it clear that the GSI-I media was able to effectively growT. pallidum in vitro.

Screening the GSI-1 media for Borrelia burgdorferi was performed by thefollowing method. Approximately 100 serum samples from clinical cases ofLyme disease were obtained. Each of the samples was inoculated in theGSI-1 media (e.g., as described in Example 2) to form a culture sample.The cultures were allowed to grow for 14 days at 37° C. Each of thecultures was then screened by phase contrast microscopy, which revealedapproximately 10-12 organisms per field. At 21 days, the culture wasexamined by direct immunofluorescent antibody staining. Examination byfluorescent microscopy demonstrated intact, motile spirochetes in eachof the samples. Examination also revealed that the number of organismsin each culture sample had increased to approximately 16-18 organismsper field. Positive results were further confirmed by PCR and dideoxyDNA sequencing (see Example 4 below). This Example makes it clear thatthe GSI-1 was able to effectively grow the Borrelia burgdorferi invitro.

Screening the GSI-1 media for Leptospira biflexa was performed by thefollowing method. Seven samples of serum from culture isolates ofpatients with multiple sclerosis and rheumatoid arthritis were obtained.Each of the seven samples were inoculated in GSI-1 (e.g., as describedin Example 2). The cultures were allowed to grow for 14 days. Each ofthe cultures was then assessed using L. biflexa specific direct antibodystaining at dilutions of 1:10, 1:20 and 1:25. The specific antibodieswere allowed to incubate for 45 minutes at 37° C. prior to counting.Examination by fluorescent microscopy demonstrated distinct, motilespirochetes in each of the seven culture samples. Examination alsorevealed that the number of organisms had increased to approximately14-18 organisms per field. This example makes it clear that GSI-1 mediawas able to effectively grow Leptospira biflexa in vitro.

Example 4 Analysis of a Borrelia burgdorferi Sample

This example describes the further analysis of a GSI-1 culture that wasidentified as Borrelia burgdorferi positive by immunofluorescentantibody staining, phase contrast microscipy, and fluorescentmicroscopy. In particular, PCR was carried out on this sample, and theamplified product was sequenced by the dideoxy sequencing method.

PCR was carried out employing the QIA Amp DNA kit (Qiagen). The twoprimers employed were: BB ASPA 56-76 5′-AAAATGTTAGCAGCCTTGACG-3′ (SEQ IDNO:1) and OSPA 438-465 5′-AGATCCATCGCTTTTAATTCCTGTGTAT-3′ (SEQ ID NO:2),both from Integrated DNA Technology Inc. These primers are specific fora 410 base pair fragment of the gene encoding the outer surface proteinA (Osp A) of Borrelia burgdorferi. The primers (each at 0.5 μl) wereadded to 20 μl of DNA isolated from the B. burgdorferi culture sample inan Eppendorf tube, along with the following reagents: 15 μl H₂O, 5 μl10×PCR Gold buffer, 4 μl of 25 mM MgCl₂, 4 μl of a dNTP mix (2.5 mM ofeach dNTP), and 1 ul of Ampli Taq Gold. The reaction conditions were asfollows: 94° C. for 3 minutes for one cycle, 35 cycles at 94° C./45seconds; 48° C./60 seconds; 72° C./1 minute; final extension 72° C./5minutes. The purified amplified PCR product was then sequenced using theCEQ 2000 DNA analysis system (Beckman). The sequencing system, whichuses the dideoxy chain termination method for sequencing, was employedas directed by the manufacturer. The determined sequence (SEQ ID NO: 3)is listed in FIG. 1. Bases 1-129 of SEQ ID NO:3 were then blastedagainst known sequences using NCBI's Blast Search program(http://www.ncbi.nlm.nih.gov/BLAST/). Three sequences representingBorrelia burgdorferi Osp A protein revealed 97% identity (128/131) withthis sequence: AF026059 (B. burgdorferi 50 kDa plasmid lipoprotein(ospA) gene, complete cds; BBT250SPA (B. burgdorferi ospA gene, T255substrain); and BBU65813 (B. burgdorferi strain Mho2 ospA gene, partialcds). These results confirm the presence of B. burgdorferi in theculture sample.

Example 5 Diagnosis of Borrelia burgdorferi Infection in a Patient

This Example describes the use of GSI-1 to diagnose Borrelia burgdorferiinfection in a human patient. The human patient experienced some form ofneurological distress for which his physicians could not determine theexact etiological agent(s). The treating physicians suspected viralencephalitis or neuroborreliosis, but a lumbar puncture test revealednothing. Consequently, a serum sample from this patient was thencultured in the GSI-1 media of the present invention using theculture/testing procedures described in Examples 2 and 3. The result ofthis testing revealed the presence of Borrelia burgdorferi in thepatient's sample. The patient was started on a course of ceftriaxone(Rocephin) and responded well until the medication was stopped.Subsequently, a brain biopsy was performed on the patient. PCRamplification specific for Borrelia burgdorferi was performed on thebiopsy sample, and the amplified product was sequenced. The resultingsequence confirmed the diagnosis of Borrelia burgdorferi infection inthe brain tissue of this patient.

Example 6 Preparation of Pellet for Antimicrobial Susceptibility Testing

This example describes the preparation of a pellet used forantimicrobial susceptibility testing of a culture sample. The procedurebegins with a culture (e.g., prepared in the manner described in Example2), where serum from a patient is inoculated in GSI-1 media and allowedto grow. In order to form pellet, an aliquot of this culture sample isplaced in a microcentrifuge tube along with 10% SDS solution, andcentrifuged at 14,000 g to disrupt the spirochetes and maximize thenumber of available eptiopic sites. The disrupted spirochetes were thencultured in an alternative growth medium made up of one part GSI-1medium, one part 6.5% lactalbumin hydrolysate, one part 2% brain heartinfusion, one part 10% pluronic fluid, and one part of the isolated(disrupted) spirochetes. This inoculated alternative growth medium wasthen allowed to incubate in the closed micro centrifuge tube until adistinctive pellet was observed (typically 7-14 days).

Example 7 Antimicrobial Susceptibility Testing

This example describes an antimicrobial susceptibility test performedfor a human patient. The patient had previously been testing using GSI-1medium (e.g., as described in Example 2) for the presence of anyspirochetes. The cultured sample revealed the presence of Treponema.This culture sample was then transferred to a microcentrifuge tube, anda pellet for antimicrobial susceptibility testing was prepared asdescribed in Example 6.

Following the observation of pellet formation, the tube was vortexed andincubated at 35° C. until a MacFarlane standard of between 8 to 12%turbidity was achieved. MICROSCAN antibiotic susceptibility microtiterplates were then inoculated with (e.g., 110 μl of) inoculum and allowedto incubate at 35° C. for 16-20 hours. The microtiter plates were thenscanned for growth and respective sensitivity reaction (both the controlwell and the growth well). Table 2 below lists the variousantimicrobials tested with this sample and whether growth of theorganism was inhibited (susceptible) or whether the growth of theorganism was not inhibited (resistant). The results in Table 2demonstrate the variable susceptibility displayed by the Treponemaisolate to various antimicrobials.

TABLE 2 Treponema Infected Patient's Antibiotic Susceptibility ResultsANTIBIOTIC TESTED RESULT Augmentin Resistant Ampicillin/SulbactamSusceptible Ampicillin Susceptible Aztreonam Susceptible CefazolinResistant Cephalothin Susceptible Ciprofloxacin Resistant GentamicinResistant Mezlocillin Susceptible Nalidixic Acid N/A NetilmicinResistant Nitrofurantoin N/A Norfloxacin N/A Piperacillin SusceptibleSulfamethoxazole N/A Tetracycline Resistant Ticarcillin/ClavulanateSusceptible Ticarcillin Susceptible Tobramycin IntermediateTrimethoprim/Sulfamethoxazole Resistant Trimethoprim N/A MeropenemResistant

Example 8 Antibiotic Susceptibility Testing of Borrelia and LeptospiraSamples

This Example describes the results for susceptibility tests that wereperformed on a large number of patient samples positive for eitherBorrelia or Leptospira. In these experiments, several hundred patientsamples were cultured on GSI-1 (e.g., as described in Example 2). Theseculture samples were then used to produce pellets for antimicrobialsusceptibility testing as described in Example 6. The antimicrobialsusceptibility tests were then carried out on each sample as describedin Example 7 or by the disc diffusion method (e.g., instead ofMICROSCAN) with the antimicrobials listed below in Table 3. Results withcontrol organisms are also provided in Table 3. The results presented inTable 3 reveal the varied response of different types of spirochetes todifferent antibiotics.

TABLE 3 Antibiotic Susceptibility Test Results Organism E. coliTatumella Y. entero gp. Borrelia Leptospira A. lwoffii Pseudomonas Totalisolates  1 2 1 519  276   1  1 Amikacin 100% 100% 100% 84% 85% 100%100% Augmentin 100% 100% 100% 90% 89% N/A N/A Ampicillin 100% 100% 100%61% 54% N/A N/A Aztreonam 100%  0%  0% 11% 15% 100% 100% Cefepime 100% 0% 100% 49% 54% 100% 100% Cefotaxime 100%  0% 100% 60% 64% 100% 100%Ceftazidime 100%  0% 100% 36% 41% 100% 100% Cefuroxime 100%  0% 100% 59%59% N/A N/A Ciprofloxacin 100% 100% 100% 84% 78% 100% 100% Gentamicin100% 100% 100% 89% 86% 100% 100% Imipenem 100% 100% 100% 84% 77% 100%100% Levofloxacin 100% 100% 100% 86% 83% 100% 100% Meropenem 100% 100%100% 82% 75% 100% 100% Piperacillin/ 100% 100% 100% 88% 86% N/A 100%Tazobactam Piperacillin 100% 100% 100% 60% 55% 100% 100% Ticarcillin/100% 100% 100% 79% 75% 100% 100% Clavulanate Tobramycin 100% 100% 100%81% 84% 100% 100%

Example 9 Susceptibility Testing of Leptospira Isolated from a Patientwith Symptoms of Multiple Sclerosis

This Example describes the results of antimicrobial susceptibilitytesting for a human patient displaying symptoms of multiple sclerosis.In particular, serum and urine samples from the patient were initiallycultured on GSI-1 (e.g., as described in Example 2), and screened forthe presence of Leptospira (e.g., as described in Example 3). Leptospirawas detected in both cultures. These cultures were then used to producepellets for antimicrobial susceptibility testing as described in Example6. The antimicrobial susceptibility tests were then carried out on eachsample with the antimicrobials and results listed below in Table 4.

TABLE 4 Antibiotic Susceptibility Results ANTIBIOTIC TESTED SERUM URINETetracycline Resistant Resistant Ofloxacin N/A N/A Ampicillin/SulbactamN/A N/A Ampicillin Resistant Resistant Rifampin Resistant ResistantClindamycin Resistant Resistant Erythromycin Resistant ResistantClarithromycin Resistant Resistant Azithromycin Resistant ResistantTrimethoprim/Sulfamethoxazole Susceptible Susceptible CefuroximeResistant Resistant Cefotaxime Resistant Resistant Ceftazidime ResistantResistant Cefpodoxime N/A N/A Amikacin Resistant Resistant GentamicinResistant Resistant Tobramycin Resistant Resistant CiprofloxacinSusceptible Susceptible Levofloxacin Susceptible Susceptible NorfloxacinN/A Susceptible Ampicillin Resistant Resistant Amoxicillin/ClavulanateResistant Resistant Piperacillin Resistant ResistantPiperacillin/Tazobactam Intermediate IntermediateTicarcillin/Clavulanate Resistant Resistant Impenem Resistant ResistantMeropenem Resistant Resistant Aztreonam Resistant Resistant

Example 10 Comparison of GSI-1 and BSK-H Media

This Example describes a comparison between GSI-1 of the presentinvention and the commercially available BSK-H. In particular, theability of each of these media to culture spirochetes from patientsamples is compared. Twenty-four patient samples were analyzed in theseexperiments, all of which were from patients previously diagnosed withLyme disease.

The procedure for culturing the patient samples on GSI-1 was carried outusing samples from clinically diagnosed Lyme patients as described inExample 2. The procedure for culturing the patient samples on BSK-H wascarried out as recommended by the manufacturer (Sigma). Briefly, BSK-Hmedium flasks were inoculated with patient samples after 0.5 to 1.0 mlof human blood or serum was added to each flask. The flasks were thenincubated between 33-35° C. for 3-4 days. The cultures were thenobserved for the presence of spirochetes. Confirmation was obtainedusing phase contrast microscopy and immunofluorescence antibodystaining.

The results of the 24 patient samples that were cultured on GSI-1revealed that 23 cultures positive for Burrelia, confirming the positivediagnosis for these patients. The only negative sample was from apatient who had undergone four years of antimicrobial treatment whichcleared the organism. Therefore, this sample was not expected to bepositive on either media. Thus, GSI-1 was successful in culturingBorrelia from 100% of the samples expected to be positive.

This data is in stark contrast to the results obtained by culturing thepatient samples on BSK-H, as only 11 of the 24 samples tested werepositive for Borrelia (i.e. only 45.8%). Furthermore, of the 13 samplesthat were negative, 12 of these samples were shown by culture on GSI-1to actually be positive for Borrelia infection (as stated above, the13th negative sample was expected to be negative). Therefore, the BSK-Hcultures had a false-negative rate of over 50% (52.2% according to theseresults). The GSI-1 media, therefore, provides a reliable media forculturing spirochetes.

Example 11 Multiple Sclerosis and Spirochete Infection

This Example describes the relationship between multiple sclerosis andspirochete infection. In particular, 146 human patients with a historyof multiple sclerosis were tested for spirochete infection. Patientsamples were collected from each patient and cultured on GSI-1 (Seee.g., Example 2 above). These samples were then tested for spirocheteinfection (e.g., as described in Example 3). The results of this testingrevealed that 146 of the 146 patient samples tested were positive forLeptospira. In other words, all of the patients with a history ofmultiple sclerosis tested for spirochete infection were found to bepositive for Leptospira spirochetes. Also, 3 of the 146 patients werealso found to be positive for Borrelia burgdorferi infection. Thesethree patients that were positive for both Leptospira and Borreliaburgdorferi were found to exhibit severe symptoms associated withmultiple sclerosis.

Scanning and transmission electron micropsy was performed onapproximately 35 of the multiple sclerosis patient samples describedabove. The electron microscopy revealed that all the Leptospiraspirochetes had a unique morphology not previously seen in otherLeptospira species. Specifically, these Leptospira were shorter inlength than other Leptospira spirochetes and tended to have a flagellarpattern characterized as having more flagella at one end than the other.

Example 12 Electron Microscopy of Rheumatoid Arthritis Patient Samples

This Example describes electron microscopy of cultivated patient samplesfrom human patients with symptoms of rheumatoid arthritis. Inparticular, several Leptospira positive patient samples from patientswith rheumatoid arthritis were examined by scanning and transmissionelectron microscopy. The electron microscopy revealed that all theLeptospira spirochetes had a unique morphology. Specifically, theseLeptospira were longer in length than other samples and had a ‘hook’structure at one end. Also, the Leptospira spirochetes were seen inclose association with mycoplasma, a phenomenon not previously reported.Specifically, the mycoplasma bacteria were commonly seen either inphysical contact with the Leptospira, or in close proximity to theLeptospira.

Example 13 Diagnosis and Treatment of a Patient with Symptoms ofRheumatoid Arthritis

This Example describes the diagnosis and treatment of a human patientwith symptoms of rheumatoid arthritis. In particular, serum and urinesamples from this patient were initially cultured on GSI-1 (e.g., asdescribed in Example 2), and screened for the presence of spirochetes(e.g., as described in Example 3). Leptospira were detected in bothsamples. These cultures were then used to produce pellets forantimicrobial susceptibility testing (e.g., as described in Example 6).The antimicrobial susceptibility tests were then carried out on eachsample (e.g., as described in Example 7) with the antimicrobials listedbelow in Table 5. The susceptibility results presented in Table 5indicate that this patient's isolate was resistant to manyantimicrobials. However, the information obtained from these assays wasused to select Ciprofloxacin and Minocycline for treatment.Ciprofloxacin (500 mg TID), Minocycline (100 mg TID), and Nystatin(500,000 units QID) were administered orally on a daily basis for over18 months without the development of resistance. After six weeks, thepatient indicated a noticeable decrease in pain and an increase in rangeof motion and energy was observed. After 6 months of therapy, thepatient experienced approximately 75-80% remission. At 18 months,cultures were still positive for spirochete, but overall numbers oforganisms were very low as compared to the initial culture.

TABLE 5 Antibiotic Susceptibility Results for Patient with Symptoms ofRheumatoid Arthritis ANTIBIOTIC TESTED SERUM URINE Trimethoprim SulfateSusceptible Susceptible Cefuroxime Resistant Resistant CefotaximeResistant Resistant Ceftazidime Resistant Resistant Cefpodoxime N/A N/AAmikacin Resistant Resistant Gentamicin Resistant Resistant TobramycinResistant Resistant Ciprofloxacin Susceptible Susceptible LevofloxacinSusceptible Susceptible Norfloxacin N/A Susceptible Ampicillin ResistantResistant Amoxicillin/Clavulanate Susceptible Susceptible PiperacillinResistant Resistant Piperacillin/Tazobactam Intermediate IntermediateTicarcillin/Clavulanate Resistant Resistant Imipenem IntermediateIntermediate Meropenem Resistant Resistant Aztreonam Resistant ResistantTetracycline Resistant Resistant Ofloxacin N/A N/A Penicillin ResistantResistant Ampicillin/Sulbactam N/A N/A Rifampin Resistant ResistantClindamycin Resistant Resistant Erythromycin Resistant ResistantClarithromycin Resistant Resistant Azithromycin Resistant Resistant

Example 14 Diagnosis and Treatment of a Patient with Symptoms ofMultiple Sclerosis

This Example describes the diagnosis and treatment of a human patientwith symptoms of multiple sclerosis. In particular, fluid samples fromthis patient were initially cultured on GSI-1 (e.g., as described inExample 2), and screened for the presence of spirochetes (e.g., asdescribed in Example 3) which revealed the presence of Leptospiraspirochetes. These cultures were then used to produce pellets forantimicrobial susceptibility testing (See e.g., Example 6), andantimicrobial susceptibility testing was carried out (See e.g., Example7). The susceptibility testing indicated the spirochete's susceptibilityto Ciprofloxacin and Minocycline. Consequently, the patient was put on acourse of daily treatment with both Ciprofloxacin and Minocycline.

The results of this treatment were dramatic. Before treatment, thispatient had many severe symptoms of multiple sclerosis includingdifficulty breathing (shallow breathing), inability to speak, andlimited upper extremities use. After 3 months of treatment, the patienthad improved breathing, regained the ability to speak, and experienceddramatic improvement in the use of upper extremities. In general,treatments of patients with symptoms of multiple sclerosis according tothe present invention have demonstrated in excess of 90% recovery overtime, with approximately 30% of affected patients showing treatmentfailures, approximately 35% showing high recovery, and approximately 35%showing 50-60% improvement.

Example 15 Diagnosing and Treatment of a Patient Suspected of Having aSpirochetal Infection

This Example describes the diagnosis and treatment of a person suspectedof having a spirochetal infection. A clinical sample is taken from aperson suffering from a neurological or autoimmune disease or otherdisease suspected of having spirochetal association. This sample is thencultured in the GSI-1 medium, as described in Example 2 and screened forspirochetal growth as described in Example 3. The patient sample isfurther screened in the alternative growth media (pellet) as describedin Example 5, and screened for antimicrobial susceptibility as describedin Example 6. A patient found to be susceptible to minocycline istreated with the following antibiotics, orally, on a daily basis:Ciprofloxacin (500 mg TID), Minocycline (100 mg TID), and Nystatin(500,000 units QID).

The response of the patient in the first 4 weeks of treatment iscontemplated to be an increase in the disease symptoms due to aJarisch-Herxheimer reaction (dexamethasone, 4 mg may be given to helpreduce the reaction), with a reduction in symptoms beginning atapproximately week 6 (increased mental clarity, increased energy, andbetter appetite). Symptoms gradually diminish over time.

Example 16 Testing Water Samples for Spirochete Contamination

This Example describes testing water samples for the presence ofspirochete contamination. In particular, water samples taken from bothupstream and downstream of a water purification facility were tested.The samples were cultured on GSI-1 as described in Example 2. Using themethods of Example 3, Leptospira were detected in both samples.

Example 17 Detection of B. burgdorferi in Maternal Serum, AmnioticFluid, and Fetal Cord Blood

This Example describes detection of B. burgdorferi in maternal serum,amniotic fluid and fetal cord blood. In particular, a mother waspreviously diagnosed with Lyme disease at least two years prior topregnancy and was treated with various antimicrobials. The attendingphysician suspected gestational Lyme disease, and therefore collectedmaternal serum, amniotic fluid, and fetal cord blood. All three sampleswere cultured on GSI-1 as described in Example 2 and Borreliaburgdorferi was detected in all three samples employing the methods inExample 3. PCR amplification specific for Borrelia burgdorferi wasperformed on all three samples, and the amplified products weresequenced. All three samples were positive for B. burgdorferi by PCRamplification, but sequence confirmation was confirmed only for thematernal serum sample and the fetal cord blood sample.

Example 18 Formulation of Reliable Spirochete Culture Media

This Example describes screening alternative media mixtures for reliablespirochete culture media. In particular, the formulation of GSI-1detailed in Example 1 is modified to create an alternative spirocheteculture media by: (1) removing one or more the components or (2)exchanging one or more of the components for components known to serve asimilar function in culture media, or (3) both removing and exchangingvarious media components. This alternative growth media is inoculatedwith a patient or water sample as described in Example 2. Standard GSI-1media is also inoculated with the sample patient or water sample forcomparison with the alternative growth spirochete culture media (i.e.,as a positive control). Alternative growth media formulations able toculture spirochetes as well (or nearly as well) as GSI-1 are selectedfor use as reliable spirochete culture media.

1. A composition comprising culture media capable of growing Treponemaorganisms in vitro under microaerophilic conditions.
 2. The compositionof claim 1, further comprising a dividing population of said Treponemaorganism.
 3. The composition of claim 1, wherein said Treponemaorganisms comprise Treponema pallidum.
 4. The composition of claim 1,wherein said culture media is serum-free culture media.
 5. Thecomposition of claim 1, wherein said culture media comprises neuralcomponents.
 6. The composition of claim 5, wherein said neuralcomponents are selected from sphingomyelin and brain extract.
 7. Thecomposition of claim 1, wherein said culture media comprises endocrinecomponents.
 8. The composition of claim 7, wherein said endocrinecomponents are selected from thyroxine, thyroid powder, and estradiol.9. The composition of claim 1, wherein said culture media comprises acell wall component.
 10. The composition of claim 9, wherein said cellwall component comprises N-acetyl glucosamine.
 11. The composition ofclaim 1, wherein said culture media comprises lactalbumin hydrolysate.12. A composition comprising serum-free culture media capable of growingTreponema, Borrelia, and Leptospira organisms.
 13. The composition ofclaim 12, further comprising a dividing population of a spirocheteorganism.
 14. The composition of claim 12, wherein said culture mediacomprises neural components.
 15. The composition of claim 14, whereinsaid neural components are selected from sphingomyelin and brainextract.
 16. The composition of claim 12, wherein said culture mediacomprises endocrine components.
 17. The composition of claim 16, whereinsaid endocrine components are selected from thyroxine, thyroid powder,and estradiol.
 18. The composition of claim 12, wherein said culturemedia comprises a cell wall component.
 19. The composition of claim 18,wherein said cell wall component comprises N-acetyl glucosamine.